Continuous variables were expressed as medians with interquartile ranges and were compared using the Mann–Whitney test. Categorical variables were expressed as numbers with percentages and compared using the chi-square test. To determine the independent predictors for survival and recovery from dialysis within 28 days after CRRT initiation, univariable and multivariable Cox proportional hazards analyses were used, and the results were presented as hazard ratios (HR) and 95% confidence intervals (CIs). Significant variables were identified through univariable analysis ( P< 0.1), and clinically important variables were considered in the multivariable analysis. Of the significant variables in the univariable analysis, those included in the SOFA or APACHE II scores i.e., mean arterial pressure, platelet count, pH, and serum creatinine were excluded from the multivariable analysis to avoid a redundant analysis. Instead, the SOFA and APACHE II scores for these variables were considered in the final multivariable analysis. I usually list out all of the items included in the set individually, but there are 55 items and we would be here all week! Payen, D. et al. Impact of continuous venovenous hemofiltration on organ failure during the early phase of severe sepsis: A randomized controlled trial. Crit. Care Med. 37(3), 803–810 (2009).

Most previous studies on fluid overload in patients with AKI receiving or not receiving RRT have included a heterogeneous population of patients with AKI, including both SIAKI and non-SIAKI 9, 10, 11, 12, 13, 14, 15, whereas only patients with SIAKI were included in the present study. To the best of our knowledge, the present study is the first to investigate the association between fluid overload and survival in patients with SIAKI receiving CRRT. Previous studies confirming the adverse effects of fluid overload on survival in patients with AKI used various definitions of the degree of fluid overload, including a percentage of fluid accumulation > 10% over the baseline weight 12, 13, 15. However, fluid overload > 10% over the baseline weight was arbitrarily defined without any basis for its definition, and the best cutoff value of the degree of fluid overload for predicting mortality was unknown. In this study, we divided fluid overload into fluid overload from AKI diagnosis to CRRT initiation (%FOpreCRRT) and total fluid overload from AKI diagnosis to ICU discharge (%FOtotal, %FOpreCRRT + %FOpostCRRT) and found that %FOpreCRRT > 4.6% (AUC, 0.826; P < 0.001) and %FOtotal > 9.6% (AUC, 0.834; P < 0.001) were the best cutoff values of the degree of fluid overload for predicting the 28-day mortality. We believe that these cutoff values could help guide fluid management in critically ill patients with SIAKI receiving CRRT and conduct further research on the association between fluid overload and survival in these patients. Marx, G. Fluid therapy in sepsis with capillary leakage. Eur. J. Anaesthesiol. 20(6), 429–442 (2003). Murugan, R. et al. Net ultrafiltration intensity and mortality in critically ill patients with fluid overload. Crit. Care 22(1), 223 (2018). CLP sepsis induces early acute kidney injury. (A) Scr had significant increases in CLP24hAKI ( n = 4) and CLP48hAKI ( n = 3) mice compared with CLPnoAKI( n = 3) mice (CLP24hAKI vs. SO, 0.19 ± 0.05 vs. 0.12 ± 0.06, p = 0.011; CLP48hAKI vs. SO, 0.21 ± 0.05 vs. 0.12 ± 0.06, p = 0.003). (B) Serum BUN had significant increases in CLPnoAKI compared with SO ( n = 3) and CLP48hAKI mice(CLPnoAKI vs. SO, 29.1 ± 9.3 vs. 18.9 ± 1.2, p = 0.039; CLPnoAKI vs. CLP48hAKI,29.1 ± 9.3 vs. 16.2 ± 7.8, p = 0.013); there are no differences among NC ( n = 3), SO, CLP24hAKI and CLP48hAKImice. NC, Normal control C57 mice; SO, Sham Operation; CLPnoAKI, CLP without AKI; CLP24hAKI, AKI after CLP 24 h; CLP48hAKIAKI after CLP 48 h. *, p<0.05; **, p<0.01; NS, p> 0.05.

Introduction

Sepsis was defined according to the American College of Chest Physicians/Society of Critical Care Medicine consensus conference criteria 18. If patients had a proven or strongly suspected bacterial infection and had at least two of the systemic inflammatory response syndrome criteria (body temperature > 38 °C or < 36 °C, heart rate > 90 bpm, respiratory rate > 20 breaths/min, PaCO 2< 32 mmHg or use of mechanical ventilation, white cell count > 12,000/mm 3 or < 4000/mm 3, or immature neutrophils > 10%), sepsis was diagnosed. AKI diagnosis was based on the Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guidelines for AKI (increase in serum creatinine ≥ 0.3 mg/dL within 48 h, increase in serum creatinine ≥ 1.5-times the baseline value, or urine volume < 0.5/kg/h for 6 h) 19. The primary outcome was the best cutoff value of fluid overload in predicting the 28-day mortality after ICU admission in the study population. The secondary outcome was a comparison of the 28-day mortality between the groups determined according to the best cutoff value of fluid overload. Fluid status assessment Increased expressions of pSTAT3 and ACE2 were associated with SIAKI. (A) Western blot analyses of STAT3, pSTAT3, Caspase 3, cleaved-caspase 3, Bcl-2 and b-actin levels in renal cortex tissues of SO and CLP mice. (B) Relative pSTAT3 levels in each group. (C) Western blot analyses of ACE2 and AGT1R expression. (D) Relative AGT1R levels in each group. (E) Relative ACE2 levels in each group; (F) Correlations of proteins expression in CLP mice. pSTAT3 level positively correlated with ACE2 expression ( R = 0.874, p< 0.001). Significant relations between 2 factors are highlighted. STAT3,signal transducer and activator of transcription 3; pSTAT3, phosphorylated STAT3; Casp3, caspase3; Clecasp3, cleaved-caspase3;Bcl-2,B-cell lymphoma-2; AGT1R, Angiotensin II Type 1 Receptor; ACE2, angiotensin converting enzyme 2. *, p<0.05; **, p<0.01; ***, p<0.001. Tolwani A. Continuous renal-replacement therapy for acute kidney injury. N Engl J Med. 2012; 367: 2505–14. pmid:23268665

Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest. 1992; 101: 1644–55. pmid:1303622 Karlsson S, Heikkinen M, Pettila V, Alila S, Vaisanen S, Pulkki K, et al. Predictive value of procalcitonin decrease in patients with severe sepsis: a prospective observational study. Crit Care. 2010; 14: R205. pmid:21078153 Tolwani, A. Continuous renal-replacement therapy for acute kidney injury. N. Engl. J. Med. 367(26), 2505–2514 (2012).All available input (intravenous fluid, blood product, total parenteral nutrition, or enteral feeding) and output (urine, drain, nasogastric tube, and stool volumes) data starting from AKI diagnosis to ICU discharge were used to assess fluid overload. The degree of cumulative fluid overload in relation to body weight was expressed as percent fluid overload (%FO), which was calculated using the following formula: ∑ daily (fluid intake in L − total output in L)/baseline body weight in kg) × 100 12. Baseline body weight was measured upon ICU admission. Based on our previous study 13, %FO was further subdivided into %FO from AKI diagnosis to CRRT initiation (%FOpreCRRT) and %FO from CRRT initiation to ICU discharge (%FOpostCRRT). Finally, total fluid overload (%FOtotal) from AKI diagnosis to ICU discharge was defined as %FOpreCRRT + %FOpostCRRT. Statistical analysis Honore PM, Redant S, De Bels D. Reliability of biomarkers of sepsis during extracorporeal therapies: the clinician needs to know what is eliminated and what is not. Crit Care. 2020; 24: 553. pmid:32917263

da Hora Passos R, Ramos JG, Mendonca EJ, Miranda EA, Dutra FR, Coelho MF, et al. A clinical score to predict mortality in septic acute kidney injury patients requiring continuous renal replacement therapy: the HELENICC score. BMC Anesthesiol. 2017; 17: 21. pmid:28173756 Fig 3. Receiver-operating characteristic curves of % PCT decrease for predicting survival (A) and recovery from dialysis (B) within 28 days after CRRT initiation in patients with SIAKI receiving CRRT. Woodward, C. W. et al. Fluid overload associates with major adverse kidney events in critically ill patients with acute kidney injury requiring continuous renal replacement therapy. Crit. Care Med. 47(9), e753–e760 (2019). Meyhoff, T. S. et al. Lower vs. higher fluid volumes during initial management of sepsis: A systematic review with meta-analysis and trial sequential analysis. Chest 157(6), 1478–1496 (2020). Our pilot CLP experiment showed that AKI mice had increased pSTAT3 and ACE2 expressions compared to SO. However, CLPAKI mice with acute tubular injury were associated with decreased PSTAT3 and ACE2 expressions. These findings suggest that STAT3 activation and increased ACE2 expression may be the compensatory response to inflammation after infection. CLPAKI mice with low response will be vulnerable to inflammatory reaction and likely to have tubular injury. S3I201 intervention experiment found that deceased pSTAT3 and ACE2 expressions due to the inhibition of STAT3 activation were not associated with SIAKI incidence, but aggravated tubular injury, which indicated that the responsive increase of pSTAT3 and ACE2 may not participate the development of SIAKI initially but play a protective role for renal tubular. The expressions of apoptosis associated proteins were no differences among SO, CLP AKI and CLP no AKI mice, which was similar to the results of a recent SIAKI study ( 31). However, increased cleaved-caspase 3 and decreased Bcl-2 expressions were detected in CLPAKI mice with BBL. Previous study also found cleaved-caspase 3 was increased in CLP rat with tubular injury ( 17).

Oh, H. J. et al. Can early initiation of continuous renal replacement therapy improve patient survival with septic acute kidney injury when enrolled in early goal-directed therapy?. J. Crit. Care 35, 51–56 (2016).

Bouchard, J. et al. Fluid accumulation, survival and recovery of kidney function in critically ill patients with acute kidney injury. Kidney Int. 76(4), 422–427 (2009). Despite its strengths, our study had some limitations. First, owing to its retrospective design, it is not possible to discern whether fluid overload is solely a marker of more severe illness or a causal contributor to mortality in our study subjects. However, as discussed above in the present study, we attempted to adjust for the disease severity indices, such as the SOFA score, APACHE II score, vasopressor use, and ventilator dependency, and found that fluid overload during CRRT was independently associated with the 28-day mortality, suggesting that fluid overload is a potentially modifiable risk factor for mortality in patients with SIAKI receiving CRRT. Second, we included a specific subset of critically ill patients, namely those with SIAKI who received CRRT. Thus, selection bias could not be avoided, and the results of our study might not be generalizable to other populations of critically ill patients with AKI. Third, fluid management using CRRT was implemented through discussion and consultation with the attending nephrologist without a standardized protocol. Thus, variations in fluid management might have affected the effect of fluid overload on survival in the present study. Uchino, S. et al. Acute renal failure in critically ill patients: A multinational, multicenter study. JAMA 294(7), 813–818 (2005).Kellum, J. A., Lameire, N., KDIGO AKI Guideline Work Group. Diagnosis, evaluation, and management of acute kidney injury: A KDIGO summary (Part 1). Critical care 17, 1–15 (2013). Baseline characteristics stratified by recovery from dialysis after CRRT initiation among survivors Increased expressions of pSTAT3 and ACE2 were associated with BBL SIAKI. (A) Western blot analyses of STAT3, pSTAT3, caspase 3, cleaved-caspase 3 and Bcl-2 levels in renal cortex tissues of CLP AKI mice. (B) Western blot analyses of ACE2 and AGT1R levels in renal cortex tissues of CLP AKI mice. (C) The differences of pSTAT3, Cleaved-caspase 3, Bcl2, AGT1R and ACE2 expressions between No BBL and BBL mice. (D) The difference of AGT1R mRNA expression determined by RT-PCT between No BBL and BBL mice. (E) The difference of ACE2 mRNA expression determined by RT-PCT between No BBL and BBL mice. *, p<0.05; **, p<0.01; ***, p<0.001.